Experiments are proposed which explore the molecular mechanisms which determine the occurrence and structure of gene amplification in methotrexate-resistant Leishmania, a protozoan parasite of humans. Gene amplification plays a significant role in oncogenesis, as well as in drug resistance observed in tumors and human pathogens. We have shown previously that in every respect gene amplification in Leishmania is similar to that observed in cultured cells where it has received most extensive study. Leishmania offers advantages in that 1) we have been able to completely isolate and characterize the amplified DNA found in the methotrexate-resistant R1000 line, initially consisting of extra-chromosomal circular DNAs from two separate chromosomal regions, and 2) the only sites of DNA rearrangement observed during amplification are the ones required to generate the amplified circles. We will examine key structural features of the amplified DNAs: first, repeated DNA sequences are invariably found near or at the site of rearrangement within the amplified circular DNAs. At least one of these regions appears at this time to resemble other eukaryotic transposable elements. We will characterize these regions by a combination of restriction mapping, DNA sequencing and electron microscopy of heteroduplexes. We will ask whether additional mutants bearing amplified DNA also contain rearrangements within the repetitive DNA regions. Second, we will isolate and characterize transcripts encoded by amplified DNA. Of specific interest is the target of methotrexate, the bifunctional dihydrofolate reductase-thymidylate synthetase characteristic of protists, which is overproduced in the amplified Leishmania. This transcript will be demonstrated by hybrid-selected translation, and this gene will be extensively characterized by cDNA cloning, blot hybridization, and DNA sequencing. Third, we shall attempt to demonstrate origins of DNA replication within the amplified DNAs by electron microscopy. Fourth, we will determine whether stably-amplified DNAs which have integrated into the chromosome have returned to the site of the resident gene or to other chromosomal regions, and if repeated DNA sequences mediate this latter process. Finally, we will examine the mutation rate to gene amplification in Leishmania, and factors which may serve to increase this rate. We intend to place these key data within the larger context of current models for the generation and properties of DNA amplification.